Investigating the Chaperone Activity of Nucleic Acids

项目摘要 这个项目旨在了解核酸如何防止蛋白质聚集和帮助蛋白质折叠。蛋白 错误折叠和聚集导致许多使人衰弱的疾病，包括阿尔茨海默氏病、帕金森氏病、 疾病和肌萎缩侧索硬化症（ALS）。除了在细胞中的许多其他角色，我们最近 发现核酸是强大的分子伴侣。监护人负责维护 蛋白质组稳定性通过防止有毒蛋白质聚集和使蛋白质折叠。鉴于 核酸在细胞中的优势，它们在应激颗粒中的作用，以及它们的有效伴侣活性， 它们很可能在蛋白质稳态中起主要作用。在目前的调查阶段， 几乎没有关于核酸如何防止蛋白质聚集，它们如何与其他蛋白质相互作用， 分子伴侣，或者它们在蛋白质折叠中扮演什么角色。这个项目将阐明核酸如何作为 分子伴侣，这些重要分子的一种以前未被认识的特性。 在K99阶段描述的工作将为我提供必要的技能，成功地 作为独立科学家完成R00阶段，并为R00阶段的研究奠定基础。在 在K99阶段，我将学习新的体外和体内技术，用于表征分子伴侣的功能， 核酸分子伴侣。使用这些技术，我将检查核酸的序列特异性， 酸伴侣使用遗传筛选（目的1A）。然后，我将生成第一个模型的基础， 核酸通过研究这些序列如何帮助 蛋白质折叠并与其他分子伴侣相互作用（Aim 1B）。我还将开发结构生物学方法， NMR光谱学和X射线晶体学来分析分子伴侣活性核苷酸的复合物 链和部分折叠的蛋白质底物（Aim 2）。 在这个项目的R00阶段，我将继续扩大我们对核酸伴侣功能的研究， 包括更大的RNA结构词汇（目标1）。我也会运用结构生物学技术 在K99阶段发展，旨在了解核酸分子伴侣结构之间的关系， 和功能，并阐明核酸分子伴侣如何影响蛋白质底物的折叠（目的2）。 总之，这些数据将使我们能够构建一个全面的模型，描述蛋白质伴侣的功能。 RNA和DNA。在未来，这种模型可能会成为设计基于核酸的治疗方法的基础。 目前无法治愈的蛋白质错误折叠疾病。

PROJECT SUMMARY This project aims to understand how nucleic acids prevent protein aggregation and aid protein folding. Protein misfolding and aggregation lead to many debilitating diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In addition to many other roles in the cell, we recently discovered that nucleic acids are powerful molecular chaperones. Chaperones are responsible for maintaining proteome stability by preventing toxic protein aggregation and enabling protein folding. Given the preponderance of nucleic acids in the cell, their role in stress granules, and their potent chaperone activity, it is highly likely that they play a major role in protein homeostasis. At the current stage of investigation, we have virtually no knowledge of how nucleic acids prevent protein aggregation, how they interact with other chaperones, or what role they play in protein folding. This project will elucidate how nucleic acids work as molecular chaperones, a previously unrecognized property of these important molecules. The work described in the K99 phase of this grant will provide me with the necessary skills to successfully complete the R00 phase as an independent scientist and will lay the foundation for the R00 phase research. In the K99 phase, I will learn new in vitro and in vivo techniques for characterizing chaperone function geared towards nucleic acid chaperones. Using these techniques, I will examine the sequence specificity of nucleic acid chaperones using a genetic screen (Aim 1A). I will then generate the foundation for the first model of how nucleic acids prevent aggregation and participate in protein folding by studying how these sequences aid in protein folding and interact with other chaperones (Aim 1B). I will also develop structural biology methods using both NMR spectroscopy and X-ray crystallography to analyze complexes of chaperone-active nucleotide chains and partially folded protein substrates (Aim 2). In the R00 phase of this project, I will continue to expand our studies of nucleic acid chaperone function to include the larger structural vocabulary of RNA (Aim 1). I will also apply the structural biology techniques developed in the K99 phase toward understanding the relationship between nucleic acid chaperone structure and function, and elucidating how nucleic acid chaperones affect the folding of protein substrates (Aim 2). Together, these data will allow us to construct a comprehensive model describing the chaperone function of RNA and DNA. In the future, this model may form the basis for designing nucleic acid-based therapeutics for currently untreatable protein misfolding diseases.